Abstract 3914: Antibodies: The key to spatial mass spectrometry success
Authors: Danielle Fails, Michael Spencer
Journal: Cancer Research (2022)

Abstract As multiplex immunofluorescent assays continue to be used more frequently than ever, their success almost exclusively relies on the quality and performance of the antibodies used. One of the most popular methods, imaging mass cytometry (IMC), primarily differs from traditional methods in that it uses 1) metal labels rather than fluorescent tags and 2) allows for the addition of all primary antibodies at one time rather than sequentially. Success in IMC, however, can be hindered by loss of antibody activity, poor optimization, or insufficient testing post-conjugation. The aim of this study was to assess whether thoroughly analyzing antibody activity before and after metal conjugation could serve as a predictor for success in IMC.Carrier-free antibodies (Bethyl Laboratories, Inc.) were conjugated using Fluidigm’s Maxpar® X8 Antibody Labelling Kits. Metal-antibody pairings were designated based on the protein’s expression level. Post-conjugation activity testing was performed on tissue microarrays using both citrate and tris HIER methods. Initial DAB testing was done first to observe any loss of signal, followed by IF testing using Dylight-conjugated secondary antibodies, to determine exposure time limitations. Initial testing concentration for each target was based off the validated working concentration provided on the data sheet for each antibody. If a loss of activity was observed, further IF testing was performed to recover activity via titration experiments. Optimal exposure times were obtained for each sample using the Vectra Polaris imaging system. The conjugates were then assessed in IMC using Fluidigm’s Hyperion System.A set of 60 antibodies were evaluated with 30% showing decreased activity post-conjugation, ranging from a minor reduction of signal to complete loss. Exposure times taken from each target were determined to be a valid predictor of success in IMC. A sliding scale showed that targets with &amp;lt;200ms exposure times would be successful while those with &amp;gt;300ms failed. Furthermore, several antibodies that showed a complete loss of signal post-conjugation were able to recover that signal via an increase in antibody concentration.The utilization of pre- and post-conjugation activity tests on metal-conjugated antibodies provides key insights on imaging mass cytometry outcomes. Exposure times from IF serve as one of the most crucial predictors for success. Additionally, the use of increased concentration gradients for first-pass “failed” antibodies can often recover any loss of activity. This loss of activity wastes time and money if not identified prior to use in imaging mass cytometry experiments. Building this sort of assessment into early IMC experiment planning can yield a streamlined approach to testing and evaluating potential IMC targets. Citation Format: Danielle Fails, Michael Spencer. Antibodies: The key to spatial mass spectrometry success [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3914.